Enhanced lipid biosynthesis via engineered plastid lipases
a technology of plastid lipases and lipids, which is applied in the direction of hydrolases, biochemistry apparatus and processes, and enzymes, can solve the problems of high environmental cost, high production cost, and high cost of petroleum exploration, and achieve enhanced expression of plastid-specific lipases, facilitate increasing oil accumulation in oil seed crops, and increase oil content
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example 1
and Methods
[0330]This Example describes some of the materials and methods employed in the development of the invention.
Plant Material and Growth Conditions
[0331]Experiments were performed with Arabidopsis thaliana ecotype Columbia (Col-0). Seeds of T-DNA insertion lines SALK_102149 (plip1-1) and SALK_147687 (plip1-2) were obtained from the Arabidopsis Biological Resource Center, Ohio State University. Lines overexpressing PLIP1 (or PLIP1S422A) were generated by subcloning the coding sequence of PLIP1 or PLIP1S422A (see below for their origin) into pEarleyGate 101 (YFP at the C-terminus) (Earley et al., 2006), followed by introducing constructs into Col-O plants by Agrobacterium tumefaciens-mediated floral dip (Clough and Bent, 1998). Transformed seeds were initially screened for resistance to Basta, followed by confirmation by RT-PCR. Primers used for genotyping of T-DNA insertion lines or for RT-PCR analysis of overexpression lines are given in Table 1. Arabidopsis seeds were verna...
example 2
a Chloroplast Thylakoid Associated Protein
[0352]The Arabidopsis genome encodes approximately 300 putative lipases (Li-Beisson et al., 2013; Troncoso-Ponce et al., 2013; Kelly and Feussner, 2016), among which 46 were included in the Chloroplast 2010 Project, aimed at assigning functions to nearly all plastid localized proteins (Lu et al., 2008; Ajjawi et al., 2010). The inventors hypothesized that some of these putative chloroplast lipases may play roles in the maintenance of photosynthetic membranes and perhaps have specialized roles in tissues with high demands on lipid metabolism such as developing seeds that accumulate TAG.
[0353]One of the predicted chloroplast lipase genes, At3g61680, encodes a protein with a conserved Lipase 3 domain and a strongly predicted transit peptide, was subsequently named PLIP1. With its Lipase 3 domain, this Arabidopsis protein has similarities to a bona fide lipase of Chlamydomonas, PGD1, involved in the turnover of chloroplast MGDG, leading to the e...
example 6
ssion of PLIP1 Increases TAG Content in Leaf Tissues
[0376]Accelerated recycling of the PG pool and exporting of 18:3 to PC only resulted in a minor increase of the amount of PC in leaves (FIG. 3), indicating that PC is an intermediate, not an end product.
[0377]To explore the ultimate fate of exported acyl groups in PLIP1-OX lines, TAG was analyzed from lyophilized whole rosettes of 4-week-old Arabidopsis plants. PLIP1-OX lines contained five to six-fold more TAG than WT and plip1 mutant lines (FIG. 3G). Labeling of PLIP1-OX lines with [14C]-acetate (FIG. 3H) confirmed that TAG labeling in PLIP1-OX1 leaves during the first day of the chase was much higher than for the EV control plants and then stabilized. Interestingly, acyl group analysis of TAG in leaves also showed a pattern of decreased 18:2 and increased 18:1 and 18:3 (FIG. 3I-3J), similar to the leaf PC acyl group composition found in PLIP1-OX lines. The trends were clearer when the ratios between 18:3 and 18:2 were calculated...
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